G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, and are the targets of a substantial fraction of all prescribed ad abused drugs. GPCRs change cellular physiology primarily by activating intracellular heterotrimeric GTP-binding proteins (G proteins). The steps involved in G protein activation include ligand binding, receptor activation, and ultimately the formation of a coupled complex between a ligand, an active receptor and an inactive G protein (LR*GGDP), leading directly to G protein activation. It has long been hypothesized that inactive GPCRs and G proteins associate with each other prior to receptor activation in precoupled or preassembled RGGDP complexes. However, the existence and relevance of these complexes in cells have been difficult to document, largely because methods to study transient interactions between membrane proteins have not been available. We recently succeeded in detecting preassembled complexes between GPCRs and Gq heterotrimers in living cells, and in demonstrating their physiological significance. However, these studies left a number of important questions unanswered, in part because they relied exclusively on ensemble measurements. One critical question is the lifetime of inactive-state preassembled receptor-G protein (RGGDP) complexes. Ensemble experiments suggest that the lifetime of a preassembled RGGDP complex is long compared to the active-state, coupled complex, but neither of these lifetimes can be determined using existing methods. Long-lived preassembled RGGDP complexes would allow receptors to self-scaffold G proteins, i.e. maintain a high local concentration of heterotrimers ready for activation. Here we propose to study RGGDP complexes using quantitative ensemble and single-molecule imaging in living cells. Ensemble imaging will allow us to determine if preassembly serves as a self-scaffolding mechanism for several different GPCRs and G protein heterotrimers. Single-molecule imaging will allow us to directly observe the formation and dissociation of inactive-state preassembled RGGDP complexes. We will thus be able to quantitatively assess the lifetimes of the macromolecular complexes important for G protein signaling. The impact of these experiments on the immediate field will be to determine if preassembly is a significant step along the pathway to G protein activation, or alternatively if itis a rare side-reaction. With respect to macromolecular interactions in general, the impact of this project will include refinement of ensemble and single-molecule imaging technology to detect membrane protein interactions in living cells, including expression systems, dyes, labeling, immobilization, imaging and analysis strategies.